Characterization of plasminogen activator in human cervical cells

Plasminogen activator activity was detected in human gynecologic specimens using a synthetic fluorogenic peptide substrate assay and confirmed by an 125I-labeled fibrin plate assay. Epithelial cells in these samples contain enzymatic activity that biochemically resembles both the well-characterized plasminogen activator, urokinase, and the less-specific plasminogen activator, trypsin. Inhibition of the cervical cell activity by diisopropylfluorophosphate and p-nitrophenyl-p'-guanidinobenzoate demonstrates that, like urokinase and trypsin, this plasminogen activator is also a serine protease. Polyacrylamide gel electrophoresis of plasminogen that had been incubated with cervical cells indicated the same mechanism of plasminogen activation as exhibited by urokinase. We attempted to correlate plasminogen activator activity of each sample with cytomorphologic diagnosis. Three of the four dysplastic samples analyzed showed higher plasminogen activator activity than did the normal samples.     

Biochemical and thrombolytic properties of a low molecular weight form (comprising Leu144 through Leu411) of recombinant single-chain urokinase-type plasminogen activator

The cDNA encoding a low Mr derivative (residues 144-411) of human single-chain urokinase-type plasminogen activator was cloned, the recombinant low Mr single-chain urokinase-type plasminogen activator (rscu-PA-32k) was expressed in Chinese hamster ovary cells, and the translation product was purified to homogeneity from conditioned cell culture medium. rscu-PA-32k is very similar to intact recombinant single-chain urokinase-type plasminogen activator in terms of its very low activity (120 IU/mg) on a chromogenic substrate for urokinase (pyroglutamylglycylarginine p-nitroanilide), its plasminogen-dependent fibrinolytic activity on fibrin plates (specific activity = 170,000 IU/mg), its plasminogen activating potential, and the lack of specific binding to fibrin. In a rabbit jugular vein thrombosis model, comparable thrombolysis was obtained with rscu-PA-32k as compared to low molecular weight two-chain urokinase (50% lysis at 2.1 and 1.6 mg/kg infused over 4 h). Thrombolysis was associated with much less extensive systemic fibrinogen breakdown with rscu-PA-32k than with two-chain urokinase (residual fibrinogen at 50% lysis of 71 and 10%, respectively). It is concluded that the functional properties of rscu-PA-32k, expressed with a high efficiency, are similar to those of its previously characterized natural counterpart.     

Plasminogen activation in diabetes mellitus. Kinetic analysis of plasmin formation using components isolated from the plasma of diabetic donors

Two components of the fibrinolytic system, plasminogen and the vascular plasminogen activator, have been isolated to apparent homogeneity from the post-venous occlusion plasma of three diabetic patients (hemoglobin A1C greater than 7%) and of one nondiabetic control person. Plasminogen activation was studied for each person separately in the absence and presence of CNBr fragments of fibrinogen. Activation of diabetic plasminogen by urokinase was not significantly altered as compared to the activation of control plasminogen. The same was found when diabetic plasminogen was activated by control vascular plasminogen activator in the presence of fibrinogen fragments but only at plasminogen concentrations below 10-30 nM; at higher substrate concentrations, however, plasminogen activation was impaired in a pattern resembling substrate inhibition. Activation of control plasminogen by diabetic vascular plasminogen activator was completely impaired in the absence of fibrinogen fragments. Addition of fibrinogen fragments stimulated plasmin formation by diabetic vascular plasminogen activator resulting in kinetic constants which were similar to the activation of control plasminogen by control vascular plasminogen activator in the absence of fibrinogen fragments (Km = 7.5 microM, kcat = 0.05 S-1). Addition of fibrinogen fragments in controls decreased Km values to less than 0.1 microM. Despite addition of fibrinogen fragments the rate of plasmin formation from diabetic plasminogen by diabetic vascular plasminogen activator isolated from the same diabetic donor was so small that kinetic constants could not be calculated.     

Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro

Tissue plasminogen activator (tPA) was covalently linked by disulfide bonds to a monoclonal antibody specific for the amino terminus of the beta chain of fibrin (antibody 59D8). The activity of the tPA-59D8 conjugate was compared with that of tPA, urokinase (UK), and a UK-59D8 conjugate. For lysis of fibrin monomer, tPA was 10 times as potent as UK, whereas both UK-59D8 and tPA-59D8 conjugates were 100 times as potent as UK and 10 times as potent as tPA. Conjugation of tPA or UK to antibody 59D8 produced a 3.2-4.5-fold enhancement in clot lysis in human plasma over that of the respective unconjugated plasminogen activator. However, the UK-59D8 conjugate was only as potent as tPA alone. Antibody-conjugated tPA or UK consumed less fibrinogen, alpha 2-antiplasmin, and plasminogen than did the unconjugated activators, at equipotent fibrinolytic concentrations. Antibody targeting thus appears to increase the concentration of tPA in the vicinity of a fibrin deposit, which thereby leads to enhanced fibrinolysis.     

Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.     

Fibrin autography of plasminogen activator by electrophoretic transfer into fibrin agar gels

An electrophoretic modification of the conventional fibrin autography that can be used for the detection of plasminogen activators (urokinase type and tissue type) and fibrin-degrading enzymes in complex biological fluids is described. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins and the substrate plasminogen are transferred electrophoretically into the fibrin indicator gel, resulting in an efficient transfer of proteinases as well as high resolution and contrast of fibrinolytic zones caused by plasminogen activator activity. Picogram amounts of human urokinase type plasminogen activator (about 0.002 International Unit) are still detectable. The technique is also applicable to reversed fibrin autography for plasminogen activator inhibitors.     

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.     

Kinetic studies on the effect of heparin and fibrin on plasminogen activators.

1. Possible interactions between fibrin(ogen) and heparin in the control of plasminogen activation were studied in model systems using the thrombolytic agents tissue-type plasminogen activator (t-PA), urokinase and streptokinase.plasminogen activator complex and the substrates Glu- and Lys-plasminogen. 2. Both t-PA and urokinase activities were promoted by heparin and by pentosan polysulphate, but not by chondroitin sulphate or hyaluronic acid. The effect was on Km. 3. In the presence of soluble fibrin (and its mimic, CNBr-digested fibrinogen) the effect of heparin on t-PA was attenuated, although not abolished. In studies using a monoclonal antibody and 6-aminohexanoic acid, it was found that heparin and fibrin did not seem to share a binding site on t-PA. 4. The activity of t-PA B-chain was unaffected by heparin, so the binding site is located on the A-chain of t-PA (and urokinase). 5. Fibrin potentiated the activity of heparin on urokinase. The activity of streptokinase.plasminogen was unaffected by heparin whether or not fibrin was present. 6. If these influences of heparin and fibrin also occur in vivo, then, in the presence of heparin, the relative fibrin enhancement of t-PA will be diminished and the likelihood of systemic activation by t-PA is increased.     

Magnetic urokinase: targeting of urokinase to fibrin clot

A plasminogen activator of human origin, urokinase, was endowed with magnetic property. The magnetic urokinase was composed of magnetite, polyethylene glycol derivative and urokinase, and dispersed in saline. Its particle size of magnetite was approximately 30-60 nm. It was selectively delivered to fibrin clot by magnetic force in continuously circulating plasma and exerted fibrinolytic activity without degrading fibrinogen.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

Isolation of urokinase by affinity ultrafiltration

A water-soluble, ligand-bound polymer has been synthesized for the purpose of isolation of urokinase, an important plasminogen activator. The affinity polymer was formed by copolymerizing N-acryloyl-m-aminobenza-midine and acrylamide in the absence of oxygen. An affinity ultrafiltration process was then developed for isolating urokinase from an artificial solution containing peroxidase and urokinase and from a crude urine source. The process yields were determined to be 86% and 49%, respectively. The recovered urokinase exhibited a specfic activity close to that of the highest commercial grade. This article also presents a new technique for assaying urokinase by coupling plasminogen with L-benzoyl arginine-p-nitroanilide (L-BAPNA), an inexpensive chromogenic substrate.     

Tissue plasminogen activator and urokinase mediate the binding of Glu-plasminogen to plasma fibrin I. Evidence for new binding sites in plasmin-degraded fibrin I

The effect of tissue plasminogen activator (TPA) or urokinase on the specific binding of human Glu-plasminogen to fibrin I formed in plasma by clotting with Reptilase was studied using 125I-plasminogen and 131I-fibrinogen. In the absence of TPA, small amounts of plasminogen were bound to fibrin I. TPA induced binding of plasminogen to plasma fibrin I that was dependent upon the concentrations of TPA and plasminogen as well as upon the time of incubation. Plasminogen binding occurred in association with fibrin clot lysis and the formation in the clot supernatant of alpha 2-plasmin inhibitor-plasmin complexes. Urokinase also induced binding of plasminogen to plasma fibrin I that was concentration- and time-dependent. The molecular form of plasminogen bound to the fibrin I plasma clot was identified as Glu-plasminogen by dodecyl sulfate-polyacrylamide gel electrophoresis and by fast performance liquid chromatography. Further studies demonstrated that fibrin I formed from fibrinogen that had been progressively degraded by plasmin-bound Glu-plasminogen. The mole ratio of plasminogen bound increased with the time of plasmin digestion. Glu-plasminogen did not bind to fibrin I formed from fibrinogen progressively digested by human leukocyte elastase, thereby demonstrating the specificity of plasmin. These studies demonstrate that plasminogen activators regulate the binding of Glu-plasminogen to fibrin I by catalyzing plasmin-mediated modifications in the fibrin substrate.     

Sensitive fluorescence assays for urokinase using synthetic peptide 4-methoxy-beta-naphthylamide substrates

Two assays for the plasminogen activator urokinase using peptide fluorogenic substrates are described. N-carbobenzoxy-glycyl-glycyl-l-arginine-4-methoxy-β-naphthylamide (CBZ-Gly-Gly-Arg-4MβNA) can be used in a direct assay that is simple, rapid, and sensitive to as little as 0.5 IU/ml urokinase. Additional sensitivity, to 0.01 IU/ml urokinase, is obtained in a second method that uses plamsinogen as the primary substrate followed by a fluorogenic substrate assay employing N-carbobenzoxy-l-alanyl-l-alanyl-l-lysine-4-methoxy-β-naphthylamide (CBZ-Ala-Ala-Lys-4MβNA) as a specific substrate for the activated plasmin. These assays are as sensitive as the best assays presently in use and are simpler to perform. In addition, these assays can readily be used for kinetic analysis of the hydrolytic activity of urokinase or other plasminogen activators.     

Proteolytic cleavage of single-chain pro-urokinase induces conformational change which follows activation of the zymogen and reduction of its high affinity for fibrin

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.     

Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells.

The effect of extracellular matrix composition on the location, amount, and activity of cell-associated urokinase-type plasminogen activator was tested using HT-1080 cells adherent to either fibronectin or vitronectin. Specific immunoprecipitation of newly synthesized urokinase indicated that cells adherent to fibronectin synthesized 2-3-fold more urokinase than cells adherent to vitronectin. Complexes of urokinase and plasminogen activator inhibitor type 1 (PAI-1) were detected in cell layers of vitronectin-adherent but not fibronectin-adherent cells. Inhibition of PAI-1 using a neutralizing monoclonal antibody resulted in a 3-fold increase in urokinase enzymatic activity on vitronectin adherent cells. Urokinase activity on fibronectin adherent cells was only slightly increased following PAI-1 neutralization. Examination of both HT-1080 and normal human fibroblast cells by immunofluorescent microscopy localized urokinase-type plasminogen activator to discrete, focal areas underneath cells adherent to vitronectin. Urokinase was not detectable by immunofluorescence on cells adherent to fibronectin. The addition of exogenous prourokinase to locate urokinase receptors on adherent HT-1080 cells indicated that the focal localization of cell-surface urokinase resulted from the clustering of urokinase receptors following adhesion to vitronectin but not fibronectin-coated substrates. These results suggest that vitronectin can contribute to the control of cell-surface plasmin activity by regulating the synthesis of urokinase and directing the localization of urokinase receptors.     

Studies on the tissue activator of plasminogen. Distribution of activator and proteolytic activity in the subcellular fractions of rabbit kidney

1. On subcellular fractionation of rabbit kidney by differential and density-gradient centrifugation, a high proportion of the tissue activator of plasminogen activity was found to be particulate and displayed sedimentation properties associated with the lysosome-rich fraction as judged biochemically by the acid-phosphatase activity. 2. The activator activity is closely associated with a latent protease whose activity is enhanced in the presence of Triton X-100 or sodium deoxycholate in the neutral pH range. Besides hydrolysing casein this protease is also capable of attacking fibrinogen at pH7·4. 3. The pH optimum for activator activity and its inhibition by -hexanoic acid (-aminocaproic acid) point to its possible similarity to urokinase, an activator of plasminogen present in the urine of most mammals.     

Separation and characterization of nonphosphorylated and serine-phosphorylated urokinase. Catalytic properties and sensitivity to plasminogen activator inhibitor type 1.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.     

Oxygen modulates the release of urokinase and plasminogen activator inhibitor-1 by retinal pigment epithelial cells

The aims of this study were to examine the effect of oxygen, in the presence or absence of exogenous growth factors, on the release of plasminogen activators and plasminogen activator inhibitor-1 by cultured human retinal pigment epithelial cells. Antigen and activity levels of urokinase, tissue plasminogen activator and plasminogen activator inhibitor were measured in conditioned media after cells were exposed to three different oxygen environments: hypoxia, normoxia and hyperoxia. Overall proteolytic balance was determined by zymography. The effects of exogenous basic fibroblast growth factor and transforming growth factor-beta were also examined. it was found that retinal pigment epithelial cells released urokinase, tissue plasminogen activator and plasminogen activator inhibitor in measurable quantities. After 48 h, urokinase levels were highest at normoxia, reaching 7.2ng/10(6) cells (+/-2.0 SEM), whereas plasminogen activator inhibitor 1 levels were highest at hyperoxia, reaching 67.5ng/10(6) cells (+/-3.7 SEM). Tissue plasminogen activator levels were minimal (<0.5ng/10(6) cells) and unaffected by both oxygen and growth factors. Overall proteolytic activity was also greatest at normoxia. Fibroblast growth factor stimulated urokinase production dose-dependently, but plasminogen activator inhibitor only minimally. Transforming growth factor-beta stimulated plasminogen activator inhibitor production dose-dependently but urokinase only at higher concentrations. These results suggest that both oxygen tension and growth factors may interact to modulate the proteolytic properties of the human retinal pigment epithelium.     

The active and the inactive plasminogen activator inhibitor from human endothelial cell conditioned medium are immunologically and functionally related to each other

In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.     

Collagen dissolution by keratinocytes requires cell surface plasminogen activation and matrix metalloproteinase activity

Matrix metalloproteinase-14 is required for degradation of fibrillar collagen by mesenchymal cells. Here we show that keratinocytes use an alternative plasminogen and matrix metalloproteinase-13-dependent pathway for dissolution of collagen fibrils. Primary keratinocytes displayed an absolute requirement for serum to dissolve collagen. Dissolution of collagen was abolished in plasminogen-depleted serum and could be restored by the exogenous addition of plasminogen. Both plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase blocked collagen dissolution, demonstrating the requirement of both plasminogen activation and matrix metalloproteinase activity for degradation. Cell surface plasmin activity was critical for the degradation process as aprotinin, but not alpha(2)-antiplasmin, prevented collagen dissolution. Keratinocytes with single deficiencies in either urokinase or tissue plasminogen activator retained the ability to dissolve collagen. However, collagen fibril dissolution was abolished in keratinocytes with a combined deficiency in both urokinase and tissue plasminogen activator. Combined, but not single, urokinase and tissue plasminogen activator deficiency also completely blocked the activation of the fibrillar collagenase, matrix metalloproteinase-13, by keratinocytes. The activation of matrix metalloproteinase-13 in normal keratinocytes was prevented by plasminogen activator inhibitor-1 and aprotinin but not by tissue inhibitor of metalloproteinase-1 and -2, suggesting that plasmin activates matrix metalloproteinase-13 directly. We propose that plasminogen activation facilitates keratinocyte-mediated collagen breakdown via the direct activation of matrix metalloproteinase-13 and possibly other fibrillar collagenases.